ABSTRACT Skeletal muscle injuries and diseases are pervasively common in patients of many backgrounds ranging from elite athletes and soldiers to the elderly. Despite the ability of skeletal muscle to remodel and repair following injury, there are still a variety of traumatic injuries (as well as congenital and acquired disorders) that result in an irrecoverable loss of muscle mass and function, termed volumetric muscle loss (VML). Moreover, the majority of muscle injuries occur at the fibers near the interface with the tendon, known as the musculotendinous junction (MTJ). Despite these facts, clinical and tissue engineering approaches to VML and MTJ regeneration are lacking. The objective of this proposal is to assess the ability of a biomaterial scaffold mimicking the collagenous composition and continuous, graded nature of native orthopedic interfaces to promote MTJ regeneration. We will take an innovative biomaterials approach using two enabling technologies: 1) we will leverage our experience with collagen-GAG (CG) scaffolds to develop a fully three-dimensional (3D) scaffold with aligned macropores mimicking the structural anisotropy of skeletal muscle and the MTJ, and 2) we will incorporate dispersed polypyrrole (PPy) microparticles throughout the scaffold to simulate the endogenous conductivity of skeletal muscle. We hypothesize that a 3D conductive scaffold with highly aligned, anisotropic pores will present an instructive microenvironment to facilitate the repair of VML injuries. We will test this hypothesis through two aims. In Aim 1 we will interrogate the combined roles of scaffold conductivity and 3D alignment on in vitro muscle-derived cell (MDC) phenotype. Bioelectrical stimuli are known to be important in organ regeneration and are integral to normal skeletal muscle function, including the cell-cell signaling that coordinates synchronous contraction. In addition to characterization of scaffold microstructural, mechanical, and conductive properties, we will assess rat MDC viability, migration, proliferation, cytoskeletal organization, and myotube formation. In Aim 2 we will assess the combined roles of scaffold conductivity and 3D alignment on in vivo repair of a rat MTJ VML defect. We will combine the directional solidification approach described above with a layering technique to fabricate graded anisotropic scaffolds with spatially-defined conductivity and mechanics toward the repair of a VML defect in a rat model of tibialis anterior MTJ injury. Repair will be evaluated using a suite of histological and functional assays. The key innovation of this work is the design of an anisotropic conductive biomaterial that incorporates spatially-stratified compositional and architectural cues, namely the inclusion of a 3D conductive milieu with an open pore, aligned microstructure that should facilitate cell infiltration and organization. This approach is likely to establish an innovative paradigm for regenerating multi-tissue interfaces. Ultimately, we believe that our strategy will enable the repair of complex VML injuries, especially those involving damage to surrounding tissues such as tendon.